The triad junction of skeletal muscle is comprised of a single invagination of the plasma membrane that plunges into the cytoplasm (the transverse-tubules or T-tubules) that is juxtaposed with two sections of the terminal cisternae of the sarcoplasmic reticulum (SR). Screening of an antibody library for novel proteins that localize to the triad junction by immunostaining identified proteins that are implicated in excitation-contraction (E-C) coupling and other aspects of Ca2+ handling in skeletal muscle. One protein identified during the screening of this library was a novel transmembrane protein called, synaptophysin-like 2 protein (Sypl2) or mitsugumin 29 (MG29).
MG29 is nearly exclusively expressed in skeletal muscle fibers, although some minor levels of expression can be resolved in the kidney, and contains four transmembrane domains that allow the protein to localize at both the transverse (T-) tubular membrane and SR membranes of the triad junction. This subcellular distribution suggest MG29 may mediate communication between the T-tubular and junctional SR membrane. The protein structure of MG29 is homologous in amino acid sequence and shares characteristic structural features with the members of the synaptophysin family of transmembrane proteins essential for neurotransmitter release.
Synaptophysin was originally identified as an abundant and highly immunogenic membrane protein of small synaptic vesicles that is also found in dense-core chromaffin and neurosecretory granules. Synaptophysin and its homologues, synaptoporin (or synaptophysin II) and pantophysin, share a common transmembrane organization, with four membrane-spanning regions and cytoplasmic amino and carboxy termini.
A unique feature of synaptophysin is that it has an oligomeric structure, leading to the proposal that synaptophysin may be a component of the fusion pore that forms during neurotransmitter release. Moreover, Alder et al. have shown that antisense oligonucleotides complementary to the synaptophysin mRNA reduce Ca2+-dependent glutamate secretion from Xenopus oocytes induced by injection of total brain mRNA. Microinjection of synaptophysin antibody into motor neurons blocked neuromuscular transmission. These data are consistent with synaptophysin being essential for neurotransmitter secretion. However, genetic approaches to identify the function of synaptophysin have not been successful; mutant mice lacking synaptophysin show a normal phenotype. This may reflect compensation by synaptoporin or other synaptophysin family members. Indeed, mice doubly deficient in synaptophysin and synaptogyrin display defects in synaptic plasticity.
Synaptophysin has been proposed to play a structural role in vesicle formation. Based on its high capacity to bind cholesterol, synaptophysin has been implicated in the generation of membrane curvature during synaptic vesicle biogenesis. Synaptophysin is also known to tightly interact with other proteins of the synaptic vesicle membrane, i.e. synaptobrevin and the vacuolar H+-ATPase. These interactions are thought to regulate exocytotic membrane fusion at the level of the SNARE complex or fusion pore formation. The latter idea is supported by studies on yeast vacuole fusion that implicate the vacuolar ATPase directly participate in membrane fusion.
The similarities between MG29 and synaptophysin prompted an investigation into whether MG29 plays an important role in modulation of membrane structures in skeletal muscle. Skeletal muscles are among the most plastic tissue in nature, and normal muscle physiology requires the formation and maintenance of the complex membrane structures. Throughout development, aging and other processes including fatigue require constant adaptations of the skeletal muscle system, thus identification and characterization of genes and proteins involved with plasticity in skeletal muscle membrane structures is essential to understand muscle physiology, as well as treating and diagnosing pathologies related to muscle dysfunction. Accordingly, there exists an ongoing need for the development of pharmaceutical modulators of muscle function for the treatment of conditions related to muscle dysfunction.